Methods for increasing oil content in plants

ABSTRACT

The invention relates to methods for increasing the oil content in plants, preferably in the seeds of plants, by expression of glycerol-3-phosphatdehydrogenases (G3PDH) from yeast, preferably from  Saccharomyces cerevisiae . The invention also relates to expression constructs for the expression of G3PDH yeast in plants, preferably in the seeds of plants, transgenic plants expressing G3PDH, and to the use of said transgenic plants in the production of foodstuffs, feed, seeds, pharmaceuticals or fine chemicals, especially in the production of oils.

RELATED APPLICATIONS

This application is a national stage application (under 35 U.S.C. 371)of PCT/EP03/04711 filed May 6, 2003 which claims benefit to Germanapplication 102 20753.4 filed May 8, 2002, German application 102 26413.9, filed Jun. 13, 2002.

FIELD OF THE INVENTION

The invention relates to methods for increasing the oil content inplants, preferably in plant seeds, by expressing yeastglycerol-3-phosphate dehydrogenases (G3PDH), preferably fromSaccharomyces cerevisiae. The invention furthermore relates toexpression constructs for expressing yeast G3PDH in plants, preferablyin plant seeds, transgenic plants expressing yeast G3PDH, and to the useof said transgenic plants for the production of food, feeds, seed,pharmaceuticals or fine chemicals, in particular for the production ofoils.

DESCRIPTION OF THE BACKGROUND

Increasing the oil content in plants and, in particular, in plant seedsis of great interest for traditional and modern plant breeding and inparticular for plant biotechnology. Owing to the increasing consumptionof vegetable oils for nutrition or industrial applications,possibilities of increasing or modifying vegetable oils are increasinglythe subject of current research (for example Töpfer et al. (1995)Science 268:681-686). Its aim is in particular increasing the fatty acidcontent in seed oils.

The fatty acids which can be obtained from the vegetable oils are alsoof particular interest. They are employed, for example, as bases forplasticizers, lubricants, surfactants, cosmetics and the like and areemployed as valuable bases in the food and feed industries. Thus, forexample, it is of particular interest to provide rapeseed oils withfatty acids with medium chain length since these are in demand inparticular in the production of surfactants.

The targeted modulation of plant metabolic pathways by recombinantmethods allows the modification of the plant metabolism in anadvantageous manner which, when using traditional breeding methods,could only be achieved after a complicated procedure or not at all.Thus, unusual fatty acids, for example specific polyunsaturated fattyacids, are only synthesized in certain plants or not at all in plantsand can therefore only be produced by expressing the relevant enzyme intransgenic plants (for example Millar et al. (2000) Trends Plant Sci5:95-101).

Triacylgylcerides and other lipids are synthesized from fatty acids.Fatty acid biosynthesis and triacylglyceride biosynthesis can beconsidered as separate biosynthetic pathways owing to thecompartmentalization, but as a single biosynthetic pathway in view ofthe end product. Lipid synthesis can be divided into twopart-mechanisms, one which might be termed “prokaryotic” and anotherwhich may be termed “eukaryotic” (Browse et al. (1986) Biochemical J235:25-31; Ohlrogge & Browse (1995) Plant Cell 7:957-970). Theprokaryotic mechanism is localized in the plastids and encompasses thebiosynthesis of the free fatty acids which are exported into thecytosol, where they enter the eukaryotic mechanism in the form of fattyacid acyl-CoA esters and are esterified with glycerol-3-phosphate (G3P)to give phosphatidic acid (PA). PA is the starting point for thesynthesis of neutral and polar lipids. The neutral lipids aresynthesized on the endoplasmic reticulum via the Kennedy pathway(voelker (1996) Genetic Engineering, Setlow (ed.) 18:111-113; Shankline& Cahoon (1998) Annu Rev Plant Physiol Plant Mol Biol 49:611-649;Frentzen (1998) Lipids 100:161-166). Besides the biosynthesis oftriacylglycerides, G3P also plays a role in glycerol synthesis (forexample for the purposes of osmoregulation and against low-temperaturestress for example).

GP3, which is essential for the synthesis, is synthesized here by thereduction of dihydroxyacetone phosphate (DHAP) by means ofglycerol-3-phosphate dehydrogenase (G3PDH), also termed dihydroxyacetonephosphate reductase. As a rule, NADH acts as reducing cosubstrate (EC1.1.1.8). A further class of glycerol-3-phosphate dehydrogenases (EC1.1.99.5) utilizes. FAD as cosubstrate. The enzymes of this classcatalyze the reaction of DHAP to G3P. In eukaryotic cells, the twoclasses of enzymes are distributed in different compartments, thosewhich are NAD-dependent being localized in the cytosol and those whichare FAD-dependent being localized in the mitochondria (for Saccharomycescerevisiae, see, for example, Larsson et al., 1998,. Yeast 14:347-357).EP-A 0 353 049 describes an NAD-independent G3PDH from Bacillus sp. InSaccharomyces cerevisiae too, an NAD-independent G3PDH is identified(Miyata K, Nagahisa M (1969) Plant Cell Physiol 10(3):635-643).

G3PDH is an essential enzyme in prokaryotes and eukaryotes which,besides having a function in lipid biosynthesis, is one of the enzymesresponsible for maintaining the cellular redox status by acting on theNAD+/NADH ratio. Deletion of the GPD2 gene in Saccharomyces cerevisiae(one of two G3PDH isoforms in this yeast) results in reduced growthunder anaerobic conditions. In addition, G3PDH appears to play a role inthe stress response of yeast mainly to osmotic stress. Deletion of theGPD1 gene in Saccharomyces cerevisiae causes hypersensitivity to sodiumchloride.

Sequences for G3PDHs have been described for insects (Drosophilamelanogaster, Drosophila virilis), plants (Arabidopsis thaliana, Cuphealanceolata), mammals (Homo sapiens, Mus musculus, Sus scrofa, Rattusnorvegicus), fish (Salmo salar, Osmerus mordax), birds (Ovis aries),amphibians (Xenopus laevis), nematodes (Caenorhabditis elegans), algaeand bacteria.

Plant cells have at least two G3PDH isoforms, a cytoplasmic isoform anda plastid isoform (Gee R W et al. (1988) Plant Physiol 86:98-103; Gee RW et al. (1988) Plant Physiol 87:379-383). In plants, the enxymaticactivity of glycerol-3-phosphate dehydrogenase was first found in potatotubors (Santora G T et al. (1979) Arch Biochem Biophys 196:403-411).Further G3PDH activities which were localized in the cytosol and theplastids were detected in other plants such as peas, maize or soya (GeeR W et al. (1988) PLANT PHYSIOL 86(1): 98-103). G3PDHs from algae suchas, for example, two plastid G3PDH isoforms and one cytosolic G3PDHisoform from Dunaliella tertiolecta have furthermore been described (GeeR et al.(1993) Plant Physiol 103(1):243-249; Gee R et al. (1989) PLANTPHYSIOL 91(1):345-351). As regards the plant G3PDH from Cuphealanceolata, it has been proposed to obtain an increased oil content or ashift in the fatty acid pattern-by overexpression in plants (WO95/06733). However, such effects have not been proven.

Bacterial G3PDHs and their function have been described (Hsu and Fox(1970) J Bacteriol 103:410-416; Bell (1974) J Bacterial 117:1065-1076).

WO 01/21820 describes the heterologous expression of a mutated E. coliG3PDH for increased stress tolerance and modification of the fatty acidcomposition in storage oils. The mutated E. coli G3PDH (gpsA2FR)exhibits a single amino acid substitution which brings about reducedinhibition via G3P. The heterologous expression of the gpsA2FR mutantleads to glycerolipids with an increased C16 fatty acid content and,accordingly, a reduced C18 fatty acid content. The modifications in thefatty acid pattern are relatively minor: an increase of 2 to 5% in the16:0 fatty acids and of 1.5 to 3.5% in the 16:3 fatty acids, and areduction in 18:2 and 18:3 fatty acids by 2 to 5% were observed. Thetotal glycerolipid content remained unaffected.

G3PDHs from yeasts (Ascomycetes) such as

-   a) Schizosaccharomyces pombe (Pidoux AL et al. (1990) Nucleic Acids    Res 18 (23): 7145; GenBank Acc.-No.: X56162; Ohmiya R et al. (1995)    Mol Microbiol 18(5):963-73; GenBank Acc.-No.: D50796, D50797),-   b) Yarrowia lipolytica (GenBank Acc.-No.: AJ250328)-   c) Zygosaccharomyces rouxii (Iwaki T et al. Yeast (2001)    18(8):737-44; GenBank Acc.-No: AB047394, AB047395, AB047397) or-   d) Saccharomyces cerevisiae (Albertyn J et al. (1994) Mol Cell Biol    14(6):4135-44; Albertyn J et al. (1992) FEBS LETT 308(2):130-132;    Merkel J R et al. (1982) Anal Biochem 122 (1):180-185; Wang H T et    al. (1994) J Bacteriol. 176(22):7091-5; Eriksson P et al. (1995) Mol    Microbiol. 17(1):95-107; GenBank Acc.-No.: U04621, X76859, Z35169).-   e) Emericella nidulans (GenBank Acc.-No.: AF228340)-   f) Debaryomyces hansenii (GenBank Acc.-No.: AF210060) are    furthermore described.

Summary of the Invention

The present invention provides, generally, methods for increasing theoil content of plants.

One embodiment of the invention is directed to methods of increasingtotal oil content in a plant organism or a tissue, organ, part, cell orpropagation material thereof, comprising expressing a transgenic yeastglycerol-3-phosphate dehydrogenase in said plant organism or in saidtissue, organ, part, cell or propagation material thereof; and selectingthe plant organism or the tissue, organ, part, cell or propagationmaterial thereof in which the total oil content in said plant organismor in said tissue, organ, part, cell or propagation material thereof, isincreased in comparison with a corresponding untransformed plantorganism or a tissue, organ, part, cell or propagation material thereof.

Another embodiment of the invention is directed to transgenic expressioncassettes comprising a nucleic acid sequence encoding a yeastglycerol-3-phosphate dehydrogenase under the control of a functionalpromoter.

Another embodiment of the invention is directed to transgenic plantorganisms or tissues, organs, parts, cells or propagation materialsthereof, comprising a recombinant yeast glycerol-3-phosphatedehydrogenase.

Another embodiment of the invention is directed to methods for theproduction of oils, fats, free fatty acids or derivatives thereof,comprising expressing a transgenic yeast glycerol-3-phosphatedehydrogenase in a plant organism or tissue, organ, part, cell orpropagation material thereof.

Other embodiments and advantages of the invention are set forth in partin the description, which follows, and in part, may be obvious from thisdescription, or may be learned from the practice of the invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Oil content in transgenic GDP1p lines.

FIG. 2. Determination of oil content in seeds of T3 generation.

FIG. 3. Determination of the G3PDH activity in control andgdp1-transformed plants.

DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide alternative methodsfor increasing the oil content in plants. We have found that this objectis achieved by the present invention.

A first subject matter of the invention comprises a method of increasingthe total oil content in a plant organism or a tissue, organ, part, cellor propagation material thereof, comprising

-   a) the transgenic expression of yeast glycerol-3-phosphate    dehydrogenase in said plant organism or in a tissue, organ, part,    cell or propagation material thereof, and-   b) the selection of plant organisms in which—in contrast to or    comparison with the starting organism—the total oil content in said    plant organism or in a tissue, organ, part, cell or propagation    material thereof is increased.

Surprisingly, it has been found that the seed-specific heterologousexpression of the yeast protein Gpd1p (G3PDH from Saccharomycescerevisiae; SEQ ID NO: 2) in Arabidopsis seeds leads to a significantlyincreased triacylglyceride (storage oils) content. The oil content wasincreased by approximately 22%, in a transgenic line even by 41%,compared with wild-type control plants (see FIG. 1). The transgenicexpression of the yeast glycerol 3-phosphate dehydrogenase had noadverse effects on the growth or other properties of the transformedplants. Since G3PDH is a biosynthetic key enzyme in all plant organisms,the method according to the invention can be applied in principle to allplant species, in addition to the species Arabidopsis thaliana, which isemployed as model plant. The method according to the invention ispreferably applied to oil crops whose oil content is already naturallyhigh and/or for the industrial production of oils.

“Plant” organism or tissue, organ, part, cell or propagation materialthereof” is generally understood as meaning any single- or multi-celledorganism or a cell, tissue, part or propagation material (such as seedsor fruit) of same which is capable of photosynthesis. Included for thepurpose of the invention are all genera and species of higher and lowerplants of the Plant Kingdom. Annual, perennial, monocotyledonous anddicotyledonous plants are preferred. Also included are mature plants,seeds, shoots and seedlings, and parts, propagation material (forexample tubors, seeds or fruits) and cultures derived from them, forexample cell cultures or callus cultures.

For the purposes of the invention, “plant” refers to all genera andspecies of higher and lower plants of the Plant Kingdom. The termincludes the mature plants, seeds, shoots and seedlings, and parts,propagation material, plant organ tissue, protoplasts, callus and othercultures, for example cell cultures, derived from them, and all otherspecies of groups of plant cells giving functional or structural units.Mature plants refers to plants at any developmental stage beyond theseedling. Seedling refers to a young, immature-plant at an earlydevelopmental stage.

“Plant” encompasses all annual and perennial monocotyldedonous ordicotyledonous plants and includes by way of example, but not bylimitation, those of the genera Cucurbita, Rosa, Vitis, Juglans,Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna,Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica,Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon,Nicotiana, Solarium, Petunia, Digitalis, Majorana, Cichorium,Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis,Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio,Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium,Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum, Picea andPopulus.

Preferred plants are those from the following plant families:Amaranthaceae, Asteraceae, Brassicaceae, Carophyllaceae, Chenopodiaceae,Compositae, Cruciferae, Cucurbitaceae, Labiatae, Leguminosae,Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceae, Rubiaceae,Saxifragaceae, Scrophulariaceae, Solanaceae, Sterculiaceae,Tetragoniaceae, Theaceae, Umbelliferae.

Preferred monocotyledonous plants are selected in particular from themonocotyledonous crop plants such as, for example, the Gramineae family,such as rice, maize, wheat or other cereal species such as barley,millet and sorghum, rye, triticale or oats, and sugar cane, and allgrass species.

The invention is applied very particularly preferably fromdicotyledonous plant organisms. Preferred dicotyledonous plants areselected in particular from the dicotyledonous crop plants such as, forexample,

-   -   Asteraceae such as sunflower, tagetes or calendula and others,    -   Compositae, especially the genus Lactuca, very particularly the        species sativa (lettuce) and others,    -   Cruciferae, particularly the genus Brassica, very particularly        the specis napus (oilseed rape), campestris (beet), oleracea cv        Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and        oleracea cv Emperor (broccoli) and other cabbages; and the genus        Arabidopsis, very particularly the species thaliana, and cress        or canola and others,    -   Cucurbitaceae such as melon, pumpkin/squash or zucchini and        others,    -   Leguminosae, particularly the genus Glycine, very particularly        the species max (soybean), soya, and alfalfa, pea, beans or        peanut and others,    -   Rubiaceae, preferably the subclass Lamiidae such as, for example        Coffea arabica or Coffea liberica (coffee bush) and others,    -   Solanaceae, particularly the genus Lycopersicon, very        particularly the species esculentum (tomato), the genus Solanum,        very particularly the species tuberosum (potato) and melongena        (aubergine) and the genus Capsicum, very particularly the genus        annuum (pepper) and tobacco or paprika and others,    -   Sterculiaceae, preferably the subclass Dilleniidae such as, for        example, Theobroma cacao (cacao bush) and others,    -   Theaceae, preferably the subclass Dilleniidae such as, for        example, Camellia sinensis or Thea sinensis (tea shrub) and        others,    -   Umbelliferae, particularly the genus Daucus (very particularly        the species carota (carrot)) and Apium (very particularly the        species graveolens dulce (celery)) and others;        and linseed, cotton, hemp, flax, cucumber, spinach, carrot,        sugar beet and the various tree, nut and grapevine species, in        particular banana and kiwi fruit.

Also encompassed are ornamental plants, useful or ornamental trees,flowers, cut flowers, shrubs or turf. Plants which may be mentioned byway of example but not by limitation are angiosperms, bryophytes suchas, for example, Hepaticae (liver flowers) and Musci (mosses);pteridophytes such as ferns, horsetail and clubmosses; gymnosperms suchas conifers, cycads, ginkgo and Gnetatae, the families of the Rosaceaesuch as rose, Ericaceae such as rhododendron and azalea, Euphorbiaceaesuch as poinsettias and croton, Caryophyllaceae such as pinks,Solanaceae such as petunias, Gesneriaceae such as African violet,Balsaminaceae such as touch-me-not, Orchidaceae such as orchids,Iridaceae such as gladioli, iris, freesia and crocus, Compositae such asmarigold, Geraniaceae such as geranium, Liliaceae such as dracena,Moraceae such as ficus, Araceae such as cheeseplant and many others.

Furthermore, plant organisms for the purposes of the invention arefurther organisms capable of being photosynthetically active such as,for example, algae, cyanobacteria and mosses. Preferred algae are greenalgae such as, for example, algae from the genus Haematococcus,Phaedactylum tricornatum, Volvox or Dunaliella. Synechocystis isparticularly preferred.

Most preferred are oil crops. Oil crops are understood as being plantswhose oil content is already naturally high and/or which can be used forthe industrial production of oils. These plants can have a high oilcontent and/or else a particular fatty acid composition which is ofinterest industrially. Preferred plants are those with a lipid contentof at least 1% by weight. Oil crops encompass by way of example: Boragoofficinalis (borage); Brassica species such as B. campestris, B. napus,B. rapa (mustard, oilseed rape or turnip rape); Cannabis sativa (hemp);Carthamus tinctorius (safflower); Cocos nucifera (coconut); Crambeabyssinica (crambe); Cuphea species (Cuphea species yield fatty acids ofmedium chain length, in particular for industrial applications); Elaeisguinensis (African oil palm); Elaeis oleifera (American oil palm);Glycine max (soybean); Gossypium hirsutum (American cotton); Gossypiumbarbadense (Egyptian cotton); Gossypium herbaceum (Asian cotton);Helianthus annuus (sunflower); Linum usitatissimum (linseed or flax);Oenothera biennis (evening primrose); Olea europaea (olive); Oryzasativa (rice); Ricinus communis (castor); Sesamum indicum (sesame);Triticum species (wheat); Zea mays (maize), and various nut species suchas, for example, walnut or almond.

“Total oil content” refers to the sum of all oils, preferably to the sumof the triacylglycerides.

“Oils” encompasses neutral and/or polar lipids and mixtures of these.Those mentioned in Table 1 may be mentioned by way of example, but notby limitation.

TABLE 1 Classes of plant lipids Neutral lipids Triacylglycerol (TAG)Diacylglycerol (DAG) Monoacylglycerol (MAG) Polar lipidsMonogalactosyldiacylglycerol (MGDG) Digalactosyldiacylglycerol (DGDG)Phosphatidylglycerol (PG) Phosphatidylcholine (PC)Phosphatidylethanolamine (PE) Phosphatidylinositol (PI)Phosphatidylserine (PS) Sulfoquinovosyldiacylglycerol

Neutral lipids preferably refers to triacylglycerides. Both neutral andpolar lipids may comprise a wide range of various fatty acids. The fattyacids mentioned in Table 2 may be mentioned by way of example, but notby limitation.

TABLE 2 Overview over various fatty acids (selection) Nomenclature¹ Name16:0 Palmitic acid 16:1 Palmitoleic acid 16:3 Roughanic acid 18:0Stearic acid 18:1 Oleic acid 18:2 Linoleic acid 18:3 Linolenic acidγ-18:3 Gamma-linolenic acid* 20:0 Arachidic acid 22:6 Docosahexanoicacid (DHA)* 20:2 Eicosadienoic acid 20:4 Arachidonic acid (AA)* 20:5Eicosapentaenoic acid (EPA)* 22:1 Erucic acid ¹Chain length: number ofdouble bonds *not naturally occurring in plants

Oils preferably relates to seed oils.

“Increase in” the total oil content refers to the increased oil contentin a plant or a part, tissue or organ thereof, preferably in the seedorgans of the plants. In this context, the oil content is at least 5%,preferably at least 10%, particularly preferably at least 15%, veryparticularly preferably at least 20%, most preferably at least 25%increased under otherwise identical conditions in comparison with astarting plant which has not been subjected to the method according tothe invention, but is otherwise unmodified. Conditions in this contextmeans all of the conditions which are relevant for germination, cultureor growth of the plant, such as soil conditions, climatic conditions,light conditions, fertilization, irrigation, plant protection treatmentand the like.

“Yeast glycerol 3-phosphate dehydrogenase” (termed “yeast G3PDH”hereinbelow) generally refers to all those enzymes which are capable ofconverting dihydroxyacetone phosphate (DHAP) into glycerol-3-phosphate(G3P)—preferably using a cosubstrate such as NADH—and which arenaturally expressed in a yeast.

Yeast refers to the group of unicellular fungi with a pronounced cellwall and formation of pseudomycelium (in contrast to molds). Theyreproduce vegetatively by budding and/or fission (Schizosaccharomycesand Saccharomycodes, respectively).

Encompassed are what are known as false yeasts, preferably the familiesCryptococcaceae, Sporobolomycetaceae with the genera Cryptococcus,Torulopsis, Pityrosporum, Brettanomyces, Candida, Kloeckera,Trigonopsis, Trichosporon, Rhodotorula and Sporobolomyces and Bullera,and true yeasts (yeasts which also reproduce sexually; ascus),preferably the families endo- and saccharomycetaceae, with the generaSaccharomyces, Debaromyces, Lipomyces, Hansenula, Endomycopsis, Pichia,Hanseniaspora. Most preferred are the genera Saccharomyces cerevisiae,Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Zygosaccharomyces rouxii, Yarrowia lipolitica,Emericella nidulans, Aspergillus nidulans, Debaryomyces hansenii andTorulaspora hansenii.

Yeast G3PDH refers in particular to polypeptides which have thefollowing characteristics as “essential characteristics”:

-   a) the conversion of dihydroxyacetone phosphate into    glycerol-3-phosphate using NADH as cosubstrate (EC 1.1.1.8), and-   b) a peptide sequence encompassing at least one sequence motif    selected from the group of sequence motifs consisting of

i) GSGNWGT(A/T)IAK (SEQ ID NO: 22) ii) CG(V/A)LSGAN(L/I/V)AXE(V/I)A (SEQID NO: 26) iii) (L/V)FXRPYFXV (SEQ ID NO: 27)

-    preferred is the sequence motif selected from the group consisting    of

iv) GSGNWGTTIAKV(V/I)AEN (SEQ ID NO: 29) v) NT(K/R)HQNVKYLP (SEQ ID NO:30) vi) D(I/V)LVFN(I/V)PHQFL (SEQ ID NO: 31) vii) RA(I/V)SCLKGFE (SEQ IDNO: 32) viii) CGALSGANLA(P/T)EVA (SEQ ID NO: 33) ix) LFHRPYFHV (SEQ IDNO: 34) x) GLGEII(K/R)FG (SEQ ID NO: 35)

-    the peptide sequence particularly preferably comprises at least 2    or 3, very particularly preferably at least 4 or 5, most preferably    all of the sequence motifs selected from the group of the sequence    motifs i), ii) and iii) or selected from the group of the sequence    motifs iv), v), vi), vii), viii), ix) and xiv). (Terms in brackets    refer to amino acids which are possible at this position as    alternatives; for example (V/I) means that valin or isoleucin are    possible at this position).

Moreover, a yeast G3PDH may optionally comprise—in addition to at leastone of the abovementioned sequence motifs i) to x)—further sequencemotifs selected from the group consisting of

(SEQ ID NO: 23) xi) H(E/Q)NVKYL (SEQ ID NO: 24) xii)(D/N)(I/V)(L/I)V(F/W)(V/N)(L/I/V)PHQF(V/L/I) (SEQ ID NO: 25) xiii)(A/G)(I/V)SC(L/I)KG (SEQ ID NO: 28) xiv)G(L/M)(L/G)E(M/I)(I/Q)(R/K/N)F(G/S/A)

Most preferably, yeast G3PDH refers to the yeast protein Gpdlp as shownin SEQ ID NO: 2 and functional equivalents or else functionallyequivalent portions of the above.

Functional equivalents refers in particular to natural or artificialmutations of the yeast protein Gpdlp as shown in SEQ ID NO: 2 andhomologous polypeptides from other yeasts which have the same essentialcharacteristics of a yeast G3PDH as defined above. Mutations encompasssubstitutions, additions, deletions, inversions or insertions of one ormore amino acid residues. Especially preferred are the polypeptidesdescribed by SEQ ID NO: 4, 5, 7, 9, 11, 12, 14, 16, 38 or 40.

The yeast G3PDH to be employed advantageously within the scope of thepresent invention can be found readily by database searches or byscreening gene or cDNA libraries using the yeast G3PDH sequence shown inSEQ ID NO: 2, which is given by way of example, or the nucleic acidsequence as shown in SEQ ID NO: 1, which encodes the latter, as searchsequence or probe.

Said functional equivalents preferably have at least 60%, particularlypreferably at least 70%, particularly preferably at least 80%, mostpreferably at least 90% homology with the protein ith the SEQ ID NO: 2.

Homology between two polypeptides is understood as meaning the identityof the amino acid sequence over the entire sequence length which iscalculated by comparison with the aid of the program algorithm GAP(Wisconsin Package Version 10.0, University of Wisconsin, GeneticsComputer Group (GCG), Madison, USA), setting the following parameters:

-   -   Gap Weight: 8 Length Weight: 2    -   Average Match: 2,912 Average Mismatch: −2,003

For example, a sequence with at least 80% homology with the sequence SEQID NO: 2 at the protein level is understood as meaning a sequence which,upon comparison with the sequence SEQ ID NO: 2 with the above programalgorithm and the above parameter set has at least 80% homology.

Functional equivalents also encompasses those proteins which are encodedby nucleic acid sequences which have at least 60%, particularlypreferably at least 70%, particularly preferably at least 80%, mostpreferably at least 90% homology with the nucleic acid sequence with theSEQ ID NO: 1.

Homology between two nucleic acid sequences is understood as meaning theidentity of the two nucleic acid sequences over the entire sequencelength which is calculated by comparison with the aid of the programalgorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin,Genetics Computer Group (GCG), Madison, USA), setting the followingparameters:

Gap Weight: 50 Length Weight: 3 Average Match: 10 Average Mismatch: 0

For example, a sequence which has at least 80% homology with thesequence SEQ ID NO: 1 at the nucleic acid level is understood as meaninga sequence which, upon comparison with the sequence SEQ ID NO: 1 withthe above program algorithm with-the above. parameter set has a homologyof at least 80%.

Functional equivalents also encompass those proteins which are encodedby nucleic acid sequences which hybridize under standard conditions witha nucleic acid sequence described by SEQ ID NO: 1, the nucleic acidsequence which is complementary thereto or parts of the above and whichhave the essential characteristics for a yeast G3PDH.

“Standard hybridization conditions” is to be understood in the broadsense, but preferably refers to stringent hybridization conditions. Suchhybridization conditions are described, for example, by Sambrook J,Fritsch EF, Maniatis T et al., in Molecular Cloning (A LaboratoryManual), 2nd edition, Cold Spring Harbor Laboratory Press, 1989, pages9.31-9.57) or in Current Protocols in Molecular Biology, John Wiley &Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the conditions during thewash step can be selected from the range of high-stringency conditions(with approximately 0.2×SSC at 50° C., preferably at 65° C.) (20×SSC:0.3 M sodium citrate, 3 M NaCl, pH 7.0). Denaturing agents such as, forexample, formamide or SDS may also be employed during hybridization. Inthe presence of 50% formamide, hybridization is preferably carried outat 42° C.

The invention furthermore relates to transgenic expression constructswhich can ensure a transgenic expression of a yeast G3PDH in a plantorganism or a tissue, organ, part, cells or propagation material of saidplant organism.

The definition given above applies to yeast G3PDH, with the transgenicexpression of a yeast G3PDH described by the sequence with the SEQ IDNO: 2 being particularly preferred.

In said transgenic expression constructs, a nucleic acid moleculeencoding a yeast G3PDH is preferably in operable linkage with at leastone genetic control element (for example a promoter) which ensuresexpression in a plant organism or a tissue, organ, part, cell orpropagation material of same.

Especially preferred are transgenic expression cassettes wherein thenucleic acid sequence encoding a glycerol-3-phosphate dehydrogenase isdescribed by

-   a) a sequence with the SEQ ID NO: 1, 3, 6, 8, 10, 13, 15, 37 or 39,    or-   b) a sequence derived from a sequence with the SEQ ID NO: 1, 3, 6,    8, 10, 13, 15, 37 or 39 in accordance with the degeneracy of the    genetic code-   c) a sequence which has at least 60% identity with the sequence with    the SEQ ID NO: 1.

Operable linkage is understood as meaning, for example, the sequentialarrangement of a promoter with the nucleic acid sequence encoding ayeast G3PDH which is to be expressed (for example the sequence as shownin SEQ ID NO: 1) and, if appropriate, further regulatory elements suchas, for example, a terminator in such a way that each of the regulatoryelements can fulfil its function when the nucleic acid sequence isexpressed recombinantly. Direct linkage in the chemical sense is notnecessarily required for this purpose. Genetic control sequences suchas, for example, enhancer sequences can also exert their function on thetarget sequence from positions which are further removed or indeed fromother DNA molecules. Preferred arrangements are those in which thenucleic acid sequence to be expressed recombinantly is positioned behindthe sequence acting as promoter so that the two sequences are linkedcovalently to each other. The distance between the promoter sequence andthe nucleic acid sequence to be expressed recombinantly is preferablyless than 200 base pairs, particularly preferably less than 100 basepairs, very particularly preferably less than 50 base pairs.

Operable linkage and a transgenic expression cassette can both beeffected by means of conventional recombination and cloning techniquesas they are described, for example, in Maniatis T, Fritsch E F andSambrook J (1989) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor (N.Y.), in Silhavy T J, Berman M Lund Enquist L W (1984) Experiments with Gene Fusions, Cold Spring HarborLaboratory, Cold Spring Harbor (N.Y.), in Ausubel F M et al. (1987)Current Protocols in Molecular Biology, Greene Publishing Assoc. andWiley Interscience and in Gelvin et al. (1990) In: Plant MolecularBiology Manual. However, further sequences which, for example, act as alinker with specific cleavage sites for restriction enzymes, or of asignal peptide, may also be positioned between the two sequences. Also,the insertion of sequences may lead to the expression of fusionproteins. Preferably, the expression cassette composed of a promoterlinked to a nucleic acid sequence to be expressed can be in avector-integrated form and can be inserted into a plant genome, forexample by transformation.

However, a transgenic expression cassette is also understood as meaningthose constructs where the nucleic acid sequence encoding a yeast G3PDHis placed behind an endogenous plant promoter in such a way that thelatter brings about the expression of the yeast G3PDH.

Promoters which are preferably introduced into the transgenic expressioncassettes are those which are operable in a plant organism or a tissue,organ, part, cell or propagation material of same. Promoters which areoperable in plant organisms is understood as meaning any promoter whichis capable of governing the expression of genes, in particular foreigngenes, in plants or plant parts, plant cells, plant tissues or plantcultures. In this context, expression may be, for example, constitutive,inducible or development-dependent.

The following are preferred:

-   a) Constitutive promoters    -   “Constitutive” promoters refers to those-promoters which ensure        expression in a large number of, preferably all, tissues over a        substantial period of plant development, preferably at all times        during plant development (Benfey et al.(1989) EMBO J        8:2195-2202). A plant promoter or promoter originating from a        plant virus is especially preferably used. The promoter of the        CaMV (cauliflower mosaic virus) 35S transcript (Franck et        al. (1980) Cell 21:285-294; Odell et al. (1985) Nature        313:810-812; Shewmaker et al. (1985) Virology 140:281-288;        Gardner et al. (1986) Plant Mol Biol 6:221- 228) or the 19S CaMV        promoter (U.S. Pat. No. 5,352,605; WO 84/02913; Benfey et        al. (1989) EMBO J 8:2195-2202) are especially preferred. Another        suitable constitutive promoter is the Rubisco small subunit        (SSU) promoter (U.S. Pat. No. 4,962,028), the leguminB promoter        (GenBank Acc. No. X03677), the promoter of the nopalin synthase        from Agrobacterium, the TR dual promoter, the OCS (octopine        synthase) promoter from Agrobacterium, the ubiquitin promoter        (Holtorf S et al. (1995) Plant Mol Biol 29:637-649), the        ubiquitin 1 promoter (Christensen et al. (1992) Plant Mol Biol        18:675-689; Bruce et al. (1989) Proc Natl Acad Sci USA        86:9692-9696), the Smas promoter, the cinnamyl alcohol        dehydrogenase promoter (U.S. Pat. No. 5,683,439), the promoters        of the vacuolar ATPase subunits, the promoter of the Arabidopsis        thaliana nitrilase-1 gene (GenBank Acc. No.: U38846, nucleotides        3862 to 5325 or else 5342) or the promoter of a proline-rich        protein from wheat (WO 91/13991), and further promoters of genes        whose constitutive expression in plants is known to the skilled        worker. The CaMV 35S promoter and the Arabidopsis thaliana        nitrilase-1 promoter are particularly preferred.-   b) Tissue-specific promoters    -   Furthermore preferred are promoters with specificities for        seeds, such as, for example, the phaseolin promoter (U.S. Pat.        No. 5,504,200; Bustos M M et al. (1989) Plant Cell        1.(9):839-53), the promoter of the 2S albumin gene (Joseffson L        G et al. (1987) J Biol Chem 262:12196- 12201), the legumine        promoter (Shirsat A et al. (1989) Mol Gen Genet 215(2):326-331),        the USP (unknown seed protein) promoter (Bäumlein H et        al. (1991) Mol Gen Genet 225(3):459-67), the napin gene promoter        (U.S. Pat. No. 5,608,152; Stalberg K et al. (1996) L Planta        199:515-519), the promoter of the sucrose binding proteins (WO        00/26388) or the legumin B4 promoter (LeB4; Bäumlein H et        al. (1991) Mol Gen Genet 225: 121-128; Bäumlein et al. (1992)        Plant Journal 2(2):233-9; Fiedler U et al. (1995) Biotechnology        (NY) 13(10);:1090f), the Arabidopsis oleosin promoter (WO        98/45461), and the Brassica Bce4 promoter (Wo 91/13980).    -   Further suitable seed-specific promoters are those of the gene        encoding high-molecular weight glutenin (HMWG), gliadin,        branching enyzme, ADP glucose pyrophosphatase (AGPase) or starch        synthase. Promoters which are furthermore preferred are those        -which permit a seed-specific expression in monocots such as        maize, barley, wheat, rye, rice and the like. The promoter of        the lpt2 or lptl gene (WO 95/15389, WO 95/23230) or the        promoters described in WO 99/16890 (promoters of the hordein        gene, the glutelin gene, the oryzin gene, the prolamin gene, the        gliadin gene, the glutelin gene, the zein gene, the casirin gene        or the secalin gene) can advantageously be employed.-   c) Chemically inducible promoters    -   The expression cassettes may also contain a chemically inducible        promoter (review article: Gatz et al. (1997) Annu Rev Plant        Physiol Plant Mol Biol 48:89-108), by means of which the        expression of the exogenous gene in the plant can be controlled        at a particular point in time. Such promoters such as, for        example, the PRP1 promoter (Ward et al. (1993) Plant Mol Biol        22:361-366), a salicylic acid-inducible promoter (WO 95/19443),        a benzenesulfonamide-inducible promoter (EP 0 388 186), a        tetracyclin-inducible promoter (Gatz et al. (1992) Plant J        2:397-404), an abscisic acid-inducible promoter EP 0 335 528) or        an ethanol-cyclohexanone-inducible promoter (WO 93/21334) can        likewise be used. Also suitable is the promoter of the        glutathione-S transferase isoform II gene (GST-II-27), which can        be activated by exogenously applied safeners such as, for        example, N,N-diallyl-2,2-dichloroacetamide (WO 93/01294) and        which is operable in a large number of tissues of both monocots        and dicots.

Particularly preferred are constitutive promoters, very particularlypreferred seed-specific promoters, in particular the napin promoter andthe USP promoter.

In addition, further promoters which make possible expression in furtherplant tissues or in other organisms such as, for example, E. colibacteria, may be linked operably with the nucleic acid sequence-to beexpressed. Suitable plant promoters are, in principle, all of theabove-described promoters.

The nucleic acid sequences present in the transgenic expressioncassettes according to the invention or transgenic vectors can be linkedoperably with further genetic control sequences besides a promoter. Theterm genetic control sequences is to be understood in the broad senseand refers to all those sequences which have an effect on theestablishment or the function of the expression cassette according tothe invention. Genetic control sequences modify, for example,transcription and translation in prokaryotic or eukaryotic organisms.The transgenic expression cassettes according to the inventionpreferably encompass a plant-specific promoter 5′-upstream of thenucleic acid sequence to be expressed recombinantly in each case and, asadditional genetic control sequence, a terminator sequence3′-downstream, and, if appropriate, further customary regulatoryelements, in each case linked operably with the nucleic acid sequence tobe expressed recombinantly.

Genetic control sequences also encompass further promoters, promoterelements or minimal promoters capable of modifying theexpression-controlling properties. Thus, genetic control sequences can,for example, bring about tissue-specific expression which isadditionally dependent on certain stress factors. Such elements are, forexample, described for water stress, abscisic acid (Lam E and Chua N H,J Biol Chem 1991; 266(26): 17131 -17135) and thermal stress (Schoffl Fet al. (1989) Mol Gen Genetics 217(2-3):246-53).

Further advantageous control sequences are, for example, in theGram-positive promoters amy and SPO2, and in the yeast or fungalpromoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.

In principle all natural promoters with their regulatory sequences likethose mentioned above may be used for the method according to theinvention. In addition, synthetic promoters may also be usedadvantageously.

Genetic control sequences further also encompass the 5′-untranslatedregions, introns or nonencoding 3′-region of genes, such as, forexample, the actin-1 intron, or the Adhl-S intron 1, 2 and 6 (forgeneral reference, see: The Maize Handbook, Chapter 116, Freeling andWalbot, Eds., Springer, N.Y. (1994)). It has been demonstrated thatthese may play a significant role in regulating gene expression. Thus,it has been demonstrated that 5′-untranslated sequences can enhance thetransient expression of heterologous genes. Translation enhancers whichmay be mentioned by way of example are the tobacco mosaic virus 5′leader sequence (Gallie et al. (1987) Nucl Acids Res 15:8693-8711) andthe like. They may furthermore promote tissue specificity (Rouster J etal. (1998) Plant J 15:435-440).

The transient expression cassette can advantageously contain one or moreof what are known as enhancer sequences in operable linkage with thepromoter, and these make possible an increased recombinant expression ofthe nucleic acid sequence. Additional advantageous sequences such asfurther regulatory elements or terminators may also be inserted at the3′ end of the nucleic acid sequences to be expressed recombinantly. Oneor more copies of the nucleic acid sequences to be expressedrecombinanly may be present in the gene construct.

Polyadenylation signals which are suitable as control sequences areplant polyadenylation signals, preferably those which correspondessentially to Agrobacterium tumefaciens T-DNA polyadenylation signals,in particular those of gene 3 of the T-DNA (octopine synthase) of the Tiplasmid pTiACHS (Gielen et al. (1984) EMBO J 3:835 et seq.) orfunctional equivalents thereof. Examples of particularly suitableterminator sequences are the OCS (octopin synthase) terminator and theNOS (nopaline synthase) terminator.

Control sequences are furthermore understood as those which makepossible homologous recombination or insertion into the genome of a hostorganism, or removal from the genome. In the case of homologousrecombination, for example, the coding sequence of the specificendogenous gene can be exchanged in a directed fashion for a sequenceencoding a dsRNA. Methods such as the cre/lox technology permit thetissue-specific, possibly inducible, removal of the expression cassettefrom the genome of the host organism (Sauer B (1998) Methods.14(4):381-92). Here, certain flanking sequences are added to the targetgene (lox sequences), and these make possible removal by means of crerecombinase at a later point in time.

A recombinant expression cassette and the recombinant vectors derivedfrom it may comprise further functional elements. The term functionalelement is to be understood in the broad sense and refers to all thoseelements which have an effect on generation, replication or function ofthe expression cassettes, vectors or transgenic organisms according tothe invention.

Examples which may be mentioned, but not by way of limitation, are:

-   a) Selection markers which confer resistance to a metabolism    inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456),    antibiotics or biocides, preferably herbicides, such as, for    example, kanamycin, G 418, bleomycin, hygromycin, or    phosphinothricin and the like. Particularly preferred selection    markers are those which confer resistance to herbicides. The    following may be mentioned by way of example: DNA sequences which    encode phosphinothricin acetyltransferases (PAT) and which    inactivate glutamine synthase inhibitors (bar and pat gene),    5-enolpyruvylshikimate-3-phosphate synthase genes (EPSP synthase    genes), which confer resistance to Glyphosate®    (N-(phosphonomethyl)glycine), the gox gene, which encodes    Glyphosate®-degrading enzyme (Glyphosate oxidoreductase), the deh    gene (encoding a dehalogenase which inactivates dalapon),    sulfonylurea- and imidazolinone-inactivating acetolactate synthases,    and bxn genes which encode nitrilase enzymes which degrade    bromoxynil, the aasa gene, which confers resistance to the    antibiotic apectinomycin, the streptomycin phosphotransferase (SPT)    gene, which permits resistance to streptomycin, the neomycin    phosphotransferase (NPTII) gene, which confers resistance to    kanamycin or geneticidin, the hygromycin phosphotransferase (HPT)    gene, which confers resistance to hygromycin, the acetolactate    synthase gene (ALS), which confers resistance to sulfonylurea    herbicides (for example mutated ALS variants with, for example, the    S4 and/or Hra mutation).-   b) Reporter genes which encode readily quantifiable proteins and    which allow the transformation efficacy or the expression site or    time to be assessed via their color or enzyme activity. Very    particularly preferred in this context are reporter proteins    (Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13(1):29-44) such    as the “green fluorescence protein” (GFP) (Sheen et al.(1995) Plant    Journal 8(5):777-784), chloramphenicol transferase, a luciferase (Ow    et al. (1986) Science 234:856-859), the aequorin gene (Prasher et    al. (1985) Biochem Biophys Res Commun 126(3):1259-1268),    β-galactosidase, with β-glucuronidase being very particularly    preferred (Jefferson et al. (1987) EMBO J 6:3901-3907).    -   c) Replication origins which allow replication of the expression        cassettes or vectors according to the invention in, for        example, E. coli. Examples which may be mentioned are ORI        (origin of DNA replication), the pBR322 ori or the P15A ori        (Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2nd        ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,        N.Y., 1989).-   d) Elements which are required for agrobacterium-mediated plant    transformation such as, for example, the right or left border of the    T-DNA, or the vir region.

To select cells which have successfully undergone homologousrecombination or else cells which have succesfully been transformed, itis generally required additionally to introduce a selectable markerwhich confers resistance to a biocide (for example a herbicide), ametabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456) oran antibiotic to the cells which have successfully undergonerecombination. The selection marker permits the selection of thetransformed cells from untransformed cells (McCormick et al. (1986)Plant Cell Reports 5:81-84).

In addition, said recombinant expression cassette or vectors maycomprise further nucleic acid sequences which do not encode a yeastG3PDH and whose recombinant expression leads to a further increase infatty acid biosynthesis (as a consequence of proOIL). By way of example,but not by limitation, this proOIL nucleic acid sequence which isadditionally expressed recombinantly can be selected from among nucleicacids encoding acetyl-COA carboxylase (ACCase), glycerol-3-phosphateacyltransferase (GPAT), lysophosphatidate acyltransferase (LPAT),diacylglycerol acyltransferase (DAGAT) and phospholipid:diacylglycerolacyltransferase (PDAT). Such sequences are known to the skilled workerand are readily accessible from databases or suitable cDNA libraries ofthe respective plants.

An expression cassette according to the invention can advantageously beintroduced into an organism or cells, tissues, organs, parts or seedsthereof (preferably into plants or plant cells, tissues, organs, partsor seeds) by using vectors in which the recombinant expression cassettesare present. The invention therefore furthermore relates to saidrecombinant vectors which encompass a recombinant expression cassettefor a yeast G3PDH.

For example, vectors may be plasmids, cosmids, phages, viruses or elseagrobacteria. The expression cassette can be introduced into the vector(preferably a plasmid vector) via a suitable restriction cleavage site.The resulting vector is first introduced into E. coli. Correctlytransformed E. coli are selected, grown, and the recombinant vector isobtained with methods known to the skilled worker. Restriction analysisand sequencing may be used for verifying the cloning step. Preferredvectors are those which make possible stable integration of theexpression cassette into the host genome.

The invention furthermore relates to transgenic plant organisms ortissues, organs, parts, cells or propagation material thereof whichcomprise a yeast G3PDH as defined above, a transgenic expressioncassette for a yeast G3PDH or a transgenic vector encompassing such anexpression cassette.

Such a transgenic plant organism is generated, for example, by means oftransformation or transfection by means of the corresponding proteins ornucleic acids. The generation of a transformed organism (or atransformed cell or tissue) requires introducing the DNA in question(for example the expression vector), RNA or protein into the host cellin question. A multiplicity of methods is available for this procedure,which is termed transformation (or transduction or transfection) (Keownet al. (1990) Methods in Enzymology 185:527-537). Thus, the DNA or RNAcan be introduced for example directly by microinjection or bybombardment with-DNA-coated microparticles. The cell may also bepermeabilized chemically, for example with polyethylene glycol, so thatthe DNA may reach the cell by diffusion. The DNA can also be carried outby protoplast fusion with other DNA-comprising units such as minicells,cells, lysosomes or liposomes. Electroporation is a further suitablemethod for introducing DNA; here, the cells are permeabilized reversiblyby an electrical pulse. Soaking plant parts in DNA solutions, and pollenor pollen tube transformation, are also possible. Such methods have beendescribed (for example in Bilang et al. (1991) Gene 100:247-250; Scheidet al. (1991) Mol Gen Genet 228:104-112; Guerche et al. (-1987) PlantScience 52:111-116; Neuhause et al. (1987) Theor Appl Genet 75:30-36;Klein et al. (1987) Nature 327:70-73; Howell et al. (1980) Science208:1265; Horsch et al.(1985) Science 227:1229-1231; DeBlock et al.(1989) Plant Physiology 91:694-701; Methods for Plant Molecular Biology(Weissbach and Weissbach, eds.) Academic Press Inc. (1988); and Methodsin Plant Molecular Biology (Schuler and Zielinski, eds.) Academic PressInc. (1989)).

In plants, the methods which have been described for transforming andregenerating plants from plant tissues or plant cells are exploited fortransient or stable transformation. Suitable methods are, in particular,protoplast transformation by polyethylene glycol-induced DNA uptake, thebiolistic method with the gene gun, what is known as the particlebombardment method, electroporation, the incubation of dry embryos inDNA-containing solution, and microinjection.

In addition to these “direct” transformation techniques, transformationmay also be effected by bacterial infection by means of Agrobacteriumtumefaciens or Agrobacterium rhizogenes and the transfer ofcorresponding recombinant Ti plasmids or Ri plasmids by or by infectionwith transgenic plant viruses. Agrobacterium-mediated transformation isbest suited to cells of dicotyledonous plants. The methods aredescribed, for example, in Horsch R B et al. (1985) Science 225: 1229f).

When agrobacteria are used, the expression cassette is to be integratedinto specific plasmids, either into a shuttle vector or into a binaryvector. If a Ti or Ri plasmid is to be used for the transformation, atleast the right border, but in most cases the right and left border, ofthe Ti or Ri plasmid T-DNA is linked to the expression cassette to beintroduced as flanking region.

Binary vectors are preferably used. Binary vectors are capable ofreplication both in E. coli and in Agrobacterium. As a rule, theycontain a selection marker gene and a linker or polylinker flanked bythe right and left T-DNA border sequence. They can be transformeddirectly into Agrobacterium (Holsters et al. (1978) Mol Gen Genet163:181-187). The selection marker gene, which is, for example, thenptII gene, which confers resistance to kanamycin, permits a selectionof transformed agrobacteria. The agrobacterium which acts as hostorganism in this case should already contain a plasmid with the virregion. The latter is required for transferring-the T-DNA to the plantcells. An agrobacterium transformed in this way can be used fortransforming plant cells. The use of T-DNA for the transformation ofplant cells has been studied intensively and described (EP 120 516;Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij. Kanters B.V., Alblasserdam, Chapter V; An et al. (1985) EMBO J 4:277-287). Variousbinary vectors, some of which are commercially available, such as, forexample, pBI101.2 or pBIN19 (Clontech Laboratories, Inc. USA), areknown.

Further promoters which are suitable for expression in plants have beendescribed (Rogers et al. (1987) Meth in Enzymol 153:253-277; Schardl etal. (1987) Gene 61:1-11; Berger et al. (1989) Proc Natl Acad Sci USA86:8402-8406).

Direct transformation techniques are suitable for any organism and celltype. In cases where DNA or RNA are injected or electroporated intoplant cells, the plasmid used need not meet any particular requirements.Simple plasmids such as those from the pUC series may be used. If intactplants are to be regenerated from the transformed cells, it is necessaryfor an additional selectable marker gene to be present on the plasmid.Stably transformed cells, i.e. those which contain the inserted DNAintegrated into the DNA of the host cell, can be selected fromuntransformed cells when a selectable marker is part of the insertedDNA. By way of example, any gene which is capable of conferringresistance to antibiotics or herbicides (such as kanamycin, G418,bleomycin, hygromycin or phosphinothricin and the like) is capable ofacting as marker (see above). Transformed cells which express such amarker gene are capable of surviving in the presence of concentrationsof such an antibiotic or herbicide which kill an untransformed wildtype. Examples are mentioned above and preferably comprise the bar gene,which confers resistance to the herbicide phosphinothricin (Rathore K Set al. (1993.) Plant Mol Biol 21(5):871-884), the nptII gene, whichconfers resistance to kanamycin, the hpt gene, which confers resistanceto hygromycin, or the EPSP gene, which confers resistance to theherbicide Glyphosate. The selection marker permits selection oftransformed cells from untransformed cells (McCormick et al. (1986)Plant Cell Reports 5:81-84). The plants obtained can be bred andhybridized in the customary manner. Two or more generations should begrown in order to ensure that the genomic integration is stable andhereditary.

The above-described methods are described, for example, in Jenes B etal.(.1993) Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1,Engineering and Utilization, edited by S D Kung and R Wu, AcademicPress, pp.128-143, and in Potrykus (1991) Annu Rev Plant Physiol PlantMolec Biol 42:205-225). The construct to be expressed is preferablycloned into a vector which is suitable for transforming Agrobacteriumtumefaciens, for example pBin19 (Bevan et al. (1984) Nucl Acids Res12:8711f).

Once a transformed plant cell has been generated, an intact plant can beobtained using methods known to the skilled worker. For example, calluscultures are used as starting material. The development of shoot androot can be induced in this as yet undifferentiated cell biomass in theknown fashion. The plantlets obtained can be planted out and used forbreeding.

The skilled worker is familiar-with such methods for regenerating plantparts and intact plants from plant cells. Methods which can be used forthis purpose are, for example, those described by Fennell et al. (1992)Plant Cell Rep. 11: 567-570; Stoeger et al (1995) Plant Cell Rep.14:273-278; Jahne et al. (1994) Theor Appl Genet 89:525-533.

“Transgenic”, for example in the case of a yeast G3PDH, refers to anucleic acid sequence, an expression cassette or a vector comprisingsaid G3PDH nucleic acid sequence or to an organism transformed with saidnucleic acid sequence, expression cassette or vector all thoseconstructs established by recombinant methods in which either

-   a) the nucleic acid sequence encoding a yeast G3PDH or-   b) a genetic control sequence, for example a promoter which is    functional in plant organisms, which is linked operably with said    nucleic acid sequence under a), or-   c) (a) and (b)    are not in their natural genetic environment or have been modified    by recombinant methods, it being possible for the modification to    be, for example, a substitution, addition, deletion, inversion or    insertion of one or more nucleotide residues. Natural genetic    environment refers to the natural chromosomal locus in the source    organism or the presence in a genomic library. In the case of a    genomic library, the natural genetic environment of the nucleic acid    sequence is preferably retained, at least to some extent. The    environment flanks the nucleic acid sequence at least on one side    and has a sequence length of at least 50 bp, preferably at least 500    bp, particularly preferably at least 1000 bp, very particularly    preferably at least 5000 bp. A naturally occurring expression    cassette, for example the naturally occurring combination of the    promoter of a gene encoding for a yeast G3PDH with the corresponding    yeast G3PDH gene, becomes a transgenic expression cassette when the    latter is modified by non-natural, synthetic (“artificial”) methods    such as, for example, a mutagenization. Such methods are described    (U.S. Pat. No. 5,565,350; WO 00/15815; see also above).

Host or starting organisms which are preferred as transgenic organismsare, above all, plants in accordance with the above definition. Includedfor the purposes of the invention are all genera and species of higherand lower plants of the Plant Kingdom, in particular plants which areused for obtaining oils, such as, for example, oilseed rape, sunflower,sesame, safflower, olive tree, soya, maize, wheat and nut species.Furthermore included are the mature plants, seed, shoots and seedlings,and parts, propagation material and cultures, for example cell cultures,derived therefrom. Mature plants refers to plants at any desireddevelopmental stage beyond the seedling stage. Seedling refers to ayoung, immature plant at an early developmental stage.

The transgenic organisms can be generated with the above-describedmethods for the transformation or transfection of organisms.

The invention furthermore relates to the use of the transgenic organismsaccording to the invention and to the cells, cell cultures, parts—suchas, for example, in the case of transgenic plant organisms roots, leavesand the like—and transgenic propagation material such as seeds or fruitswhich are derived therefrom for the production of foodstuffs orfeedstuffs, pharmaceuticals or fine chemicals, in particular oils, fats,fatty acids or derivatives of these.

Besides influencing the oil content, the transgenic expression of ayeast G3PDH in plants may mediate yet further advantageous effects suchas, for example, an increased stress resistance to, for example, osmoticstress. Via increased glycerol levels, the yeast G3PDH confersprotection against this type of stress, with glycerol acting asosmoprotective substance. Such osmotic stress occurs for example insaline soils and water and is an increasing problem in agriculture.Increased stress tolerance makes it possible, for example, to use areasin which conventional arable plants are not capable of thriving foragricultural usage.

Furthermore, recombinant expression of the yeast G3PDH can influence theNADH level and thus the redox balance in the plant organism. Stress suchas, for example, drought, high or low temperatures, UV light and thelike can lead to increased NADH levels and to an increased formation ofreactive oxygen (RO). Transgenic expression of the yeast G3PDH can breakdown excessive NADH, which accumulates under said stress conditions, andthus stabilize the redox balance and alleviate the effects of thestress.

Sequences

-   1. SEQ ID NO: 1    -   Nucleic acid sequence encoding Saccharomyces cerevisiae G3PDH        (Gpd1p)-   2. SEQ ID NO: 2    -   Protein sequence encoding Saccharomyces cerevisiae G3PDH (Gpd1p)-   3. SEQ ID NO: 3    -   Nucleic acid sequence encoding Saccharomyces cerevisiae G3PDH        (Gpd2p)-   4. SEQ ID NO: 4    -   Protein sequence encoding Saccharomyces cerevisiae G3PDH (Gpd2p)-   5. SEQ ID NO: 5    -   Protein sequence encoding Saccharomyces cerevisiae G3PDH (Gpd2p)        with second alternative start codon-   6. SEQ ID NO: 6    -   Nucleic acid sequence encoding Schizosaccharomyces pombe G3PDH-   7. SEQ ID NO: 7    -   Protein sequence encoding Schizosaccharomyces pombe G3PDHD-   8. SEQ ID NO: 8    -   Nucleic acid sequence encoding Schizosaccharomyces pombe G3PDH-   9. SEQ ID NO: 9    -   Protein sequence encoding Schizosaccharomyces pombe G3PDH-   10. SEQ ID NO: 10    -   Nucleic acid sequence encoding Yarrowinia lipolytica G3PDH-   11. SEQ ID NO: 11    -   Protein sequence encoding Yarrowinia lipolytica G3PDH-   12. SEQ ID NO: 12    -   Protein sequence encoding Yarrowinia lipolytica G3PDH, with        second alternative start codon-   13. SEQ ID NO: 13    -   Nucleic acid sequence encoding Zygosaccharomyces rouxii G3PDH-   14. SEQ ID NO: 14    -   Protein sequence encoding Zygosaccharomyces rouxii G3PDH-   15. SEQ ID NO: 15    -   Nucleic acid sequence encoding Zygosaccharomyces rouxii G3PDH-   16. SEQ ID NO: 16    -   Protein sequence encoding Zygosaccharomyces rouxii G3PDH-   17. SEQ ID NO: 16    -   Expression vector based on pSUN-USP for S. cerevisiae G3PDH        (Gpd1p; 1017-2190 bp insert)

18. SEQ ID NO: 18 Oligonucleotide primer ONP15′-ACTAGTATGTCTGCTGCTGCTGATAG-3′ 19. SEQ ID NO: 19 Oligonucleotideprimer ONP2 5′-CTCGAGATCTTCATGTAGATCTAATT-3′ 20. SEQ ID NO: 20Oligonucleotide primer ONP3 5′-GCGGCCGCCATGTCTGCTGCTGCTGATAG-3′ 21. SEQID NO: 21 Oligonucleotide primer ONP4 5′-GCGGCCGCATCTTCATGTAGATCTAATT-3′

-   22-35: SEQ ID NP 22 to 35: Sequence motifs for yeast G3PDHs;

possible sequence variations are given. The variations of an individualmotif may occur in each case alone, but also in the differentcombinations with each other.

-   36. SEQ ID NO: 36    -   Expression vector pGPTV-gpd1 based on pGPTV-napin for S.        cerevisiae G3PDH (Gpd1p; gdp1 insert of 11962-13137 bp; nos        terminator: 13154-13408; napin promoter: 10807-11951).-   37. SEQ ID NO: 37    -   Nucleic acid sequence encoding Emericella nidulans G3PDH-   38. SEQ ID NO: 38    -   Amino acid encoding Emericella nidulans G3PDH-   39. SEQ ID NO: 39    -   Nucleic acid sequence encoding Debaryomyces hansenii G3PDH        (partial)-   40. SEQ ID NO: 40    -   Amino acid encoding Debaryomyces hansenii G3PDH (partial)

FIGURES

FIG. 1: Oil content in transgenic GPD1p lines

-   -   Measurement of the TAG content in T2 seeds of transgenic        Arabidopsis lines with the Saccharomyces cerevisiae Gpd1p gene        (G2 to G30). The content in corresponding untransformed plants        (wild-type plants; W1 to W10) has been determined for        comparison. 8 Arabidopsis lines with a significantly increased        oil content were identified. The error deviation stated is the        result of 3 independent measurements in each case.

FIG. 2: Determination of the oil content in seeds of the T3 generation

-   -   The data shown are the oil content (in mg lipid per g dry matter        (DM)) of individual Arabidopsis lines. Each column represents        the mean of 6 individual plants per independent line. Each plant        was analysed in triplicate. The error bars denote the standard        deviation over all values. The control plants are identified by        “col”. The numerical values of the individual data are        additionally shown in the following table (the control was set        as 100% oil content):

Rel. Oil content increase in Lines (mg/g) STD % col 278.1 12.2 100 #11304.6 18.3 110 #12 301.4 19.0 108 #13 275.2 89.7 99 #21 323.2 77.0 116#24 268.9 15.1 97 #25 293.6 23.0 106 #27 285.6 18.4 103 #41 316.1 19.1114 #53 260.3 16.4 94 #67 292.0 13.8 105 #71 244.1 11.6 88 #82 295.616.8 106

-   -   Lines with a statistically significantly increased lipid content        (lines #11, #21, #41 and #67) are presented as a black bar.

FIG. 3: Determination of the G3PDH activity in the control (“col”) andthe gdpl-transformed plants.

-   -   The G3PDG activity of the individual lines was determined as        decribed in Example 8 and is shown in nmol G3P per minute per g        of fresh weight (FW).

G3PDH Activity STD col 6.68337432 0.71785229 #11 11.8958635 1.67941604#12 9.14226124 2.25411878 #13 8.8210768 2.19519777 #21 9.884354441.04798566 #24 5.89378595 1.26005769 #25 5.14179348 1.22845409 #276.77303725 3.22220935 #41 20.8325636 5.42018531 #53 7.457949472.25573816 #67 12.7670015 0.74678353 #71 9.04748534 1.59829185 #829.37260033 2.1356558

-   -   Lines with a statistically significantly increased G3PDH        activity (lines #11, #21, #41 and #67) are presented as a black        bar. It can be seen that an increased G3PDG activity correlates        with an increased lipid content.

EXAMPLES General Methods

Unless otherwise specified, all chemicals were from Fluka (Buchs), Merck(Darmstadt), Roth (Karlsruhe), Serva (Heidelberg) and Sigma(Deisenhofen). Restriction enzymes, DNA-modifying enzymes and molecularbiological kits were from Amersham-Pharmacia (Freiburg), Biometra(Gbttingen), Roche (Mannheim), New England Biolabs (Schwalbach), Novagen(Madison, Wis., USA), Perkin-Elmer (Weiterstadt), Qiagen (Hilden),Stratagen (Amsterdam, Netherlands), Invitrogen (Karlsruhe) and Ambion(Cambridgeshire, United Kingdom). The reagents used were employed inaccordance with the manufacturer's instructions.

For example, oligonucleotides can be synthesized chemically in the knownmanner using the phosphoamidite method (Voet, Voet, 2nd edition, WileyPress New York, pages 896-897). The cloning steps carried out for thepurposes of the present invention such as, for example, restrictioncleavages, agarose gel electrophoreses, purification of DNA fragments,transfer of nucleic acids to nitrocellulose and nylon membranes, linkingDNA fragments, transformation of E. coli cells, bacterial cultures,multiplication of phages and sequence analysis of recombinant DNA, arecarried out as decribed by Sambrook et al. (1989) Cold Spring HarborLaboratory Press; ISBN 0-87969-309-6. Recombinant DNA molecules weresequenced using an ABI laser fluorescence DNA sequencer following themethod of Sanger (Sanger et al. (1977) Proc Natl Acad Sci USA74:5463-5467).

Example 1 General Methods

The plant Arabidopsis thaliana belongs to the higher plants (floweringplants). This plant is closely related to other plant species from-theCruciferae family such as, for example, Brassica napus, but also toother families of dicotyledonous plants. Owing to the high degree ofhomology of its DNA sequences or its polypeptide sequences, Arabidopsisthaliana can be employed as model plant for other plant species.

-   a) Culture of Arabidopsis plants    -   The plants are grown either on Murashige-Skoog medium        supplemented with 0.5% sucrose (Ogas et al. (1997) Science        277:91-94) or in soil (Focks & Benning (1998) Plant Physiol        118:91-101). To achieve uniform germination and flowering times,        the seeds are first placed on medium or scattered on the soil        and then stratified for two days at 4° C. After flowering, the        pods are labeled. According to the labels, pods aged 6 to 20        days post-anthesis are then harvested.

Example 2 Cloning the Yeast Gpd1 Gene

Genomic DNA from Saccharomyces cerevisiae strain S288C (Mat alpha. SUC2mal mel gal2 CUP1 flo1 flo8-1; Invitrogen, Karlsruhe, Germany) wasisolated following the protocol described hereinbelow:

A 100 ml culture was grown at 30° C. to an optical density of 1.0. 60 mlof the culture were spun down for 3 minutes at 3000×g. The pellet wasresuspended in 6 ml of twice-distilled H₂O and the suspension wasdivided between 1.5 ml containers and spun down, and the supernatant wasdiscarded. The pellets were resuspended in 200 μl of solution A, 200 μlphenol/chloroform (1:1) and 0.3 g of glass beads by vortexing and thenlysed. After addition of 200 μl of TE buffer, pH 8.0, the lysates werespun for 5 minutes. The supernatant was subjected to ethanolprecipitation with 1 ml of ethanol. After the precipitation, theresulting pellet was dissolved in 400 μl of TE buffer pH 8.0+30 μg/mlRNase A. Following incubation for 5 minutes at 37° C., 18 μl 3 M sodiumacetate solution pH 4.8 and 1 ml of ethanol were added, and theprecipitated DNA was pelleted by spinning. The DNA pellet was dissolvedin 25 μl of twice-distilled H₂O. The concentration of the genomic DNAwas determined by its absorption at 260 nm.

Solution A:

-   2% Trition-X100-   1% SDS-   0.1 M NaCl-   0.01 M Tris-HCl pH 8.0-   0.001 M EDTA

To clone the Gpd1 gene, the yeast DNA which has been isolated wasemployed in a PCR reaction with the oligonucleotide primers ONP1 andONP2.

ONP1: 5′-ACTAGTATGTCTGCTGCTGCTGATAG-3′ (SEQ ID NO: 18) ONP2:5′-CTCGAGATCTTCATGTAGATCTAATT-3′ (SEQ ID NO: 19)

Composition of the PCR reaction (50 μl):

-   5.00 μl 5 μg genomic yeast-DNA-   5.00 μl 10× buffer (Advantage polymerase)+25 mM MgCl₂-   5.00 μl 2 mM dNTP-   1.25 μl each primer (10 pmol/uL)-   0.50 μl Advantage polymerase

The Advantage polymerase employed was from Clontech.

PCR-Program:

Initial denaturation for 2 min at 95° C., then 35 cycles of 45 sec at95° C., 45 sec at 55° C. and 2 min at 72° C. Final extension for 5 minat 72° C.

The PCR products were cloned into the vector pCR2.1-TOPO (Invitrogen)following the manufacturer's instructions, resulting in the vectorpCR2.1-gpd1, and the sequence was verified by sequencing.

Cloning into the agro transformation vector PGPTV involved incubating0.5 μg of the vector pCR2.1-gpd1 with the restriction enzyme XhoI (NewEngland Biolabs) for 2 hours and subsequent incubation for 15 minuteswith Klenow fragment (New England Biolabs). After incubation for 2 hourswith SpeI, the DNA fragments were separated by gel electrophoresis. The1185 bp segment of the gpdl sequence next to the vector (3.9 kb) wasexcized from the gel, purified with the “Gel Purification” kit fromQiagen following the manufacturer's instructions and eluted with 50 μlof elution buffer. 0.1 μg of the vector PGPTV was first digested for 1hour with the restriction enzyme SacI and then incubated for 15 minuteswith Klenow fragment (New England Biolabs). 10 μl of the eluate of thegpdl fragments and 10 ng of the treated pGPTV vector were ligatedovernight at 16° C. (T4 ligase, New England Biolabs). The ligationproducts were then transformed into TOP10 cells (Stratagene) followingthe manufacturer's instructions and suitably selected, resulting in thevector pGPTV-gpd1. Positive clones are verified by sequencing and PCRusing the primers ONP1 and ONP2.

To generate the vector pSUN-USP-gpd1, a PCR was carried out with thevector pCR2.1-gpd1 using the primers ONP3 and ONP4.

(SEQ ID NO: 20) ONP3: 5′-GCGGCCGCCATGTCTGCTGCTGCTGATAG-3′ (SEQ ID NO:21) ONP4: 5′-GCGGCCGCATCTTCATGTAGATCTAATT-3′

Composition of the PCR reaction (50 μl):

-   5 ng DNA plasmid pCR2.1-gpd1-   5.00 μl 10× buffer (Advantage polymerase)+25 mM MgCl₂-   5.00 μl 2 mM dNTP-   1.25 μl each primer (10 pmol/uL)-   0.50 μl Advantage polymerase

The Advantage polymerase employed was from Clontech.

PCR-Program:

Initial denaturation for 2 min at 95° C., then 35 cycles of 45 sec at95° C., 45 sec at 55° C. and 2 min at 72° C. Final extension for 5 minat 72° C.

The 1190 bp PCR product was digested for 24 hours with the restrictionenzyme NotI. The vector pSUN-USP was digested for 2 hours with NotI andthen incubated for 15 minutes with alkaline phosphatase (New EnglandBiolabs). 100 ng of the pretreated gpdl fragment and 10 ng of thetreated vector PGPTV were ligated overnight at 16° C. (T4 Ligase fromNew England Biolabs). The ligation products were then transformed intoTOP10 cells (Stratagene) following the manufacturer's instructions andsuitably selected, resulting in the vector pSUN-USP-gpd1. Positiveclones are verified by sequencing and PCR using the primers ONP3 andONP4.

Example 3 Plasmids for the Transformation of Plants

Binary vectors such as pBinAR can be used for the transformation ofplants (Höfgen und Willmitzer (1990) Plant Science 66: 221-230). Thebinary vectors can be constructed by ligating the cDNA into T-DNA insense and antisense orientation. 5′ of the cDNA, a plant promoteractivates the transcription of the cDNA. A polyadenylation sequence islocated 3′ of the cDNA.

Tissue-specific expression can be achieved using a tissue-specificpromoter. For example, seed-specific expression can be achieved bycloning in the napin or the LeB4- or the USP promoter 5′ of the cDNA.Any other seed-specific promoter element can also be used. The CaMV 35Spromoter can be used for constitutive expression in the whole plant.

A further example of binary vectors is the vector pSUN-USP andpGPTV-napin, into which the fragment of Example 2 was cloned. The vectorpSUN-USP contains the USP promoter and the OCS terminator. The vectorpGPTV-napin contains a truncated version of the napin promoter, and thenos terminator.

The fragments of Example 2 were cloned into the multiple cloning site ofthe vector pSUN-USP and pGPTV-napin respectively, to make possible theseed-specific expression of the gdpl gene. The corresponding constructpSUN-USP-gpd1 is described with the SEQ ID NO: 17, and the construct ofG3PDH in pGPTV-napin (pGPTV-gpd1) by SEQ ID NO: 36.

Example 4 Transformation of Agrobacterium

Agrobacterium-mediated plant transformation can be carried out forexample using the Agrobacterium tumefaciens strains GV3101 (pMP90)(Koncz und Schell (1986) Mol Gen Genet 204: 383-396) or LBA4404(Clontech). Standard transformation techniques may be used for thetransformation (Deblaere et al.(1984) Nucl Acids Res 13:4777-4788).

Example 5 Transformation of Plants

Agrobacterium-mediated plant transformation can be effected usingstandard transformation and regeneration techniques (Gelvin S B,Schilperoort R, Plant Molecular Biology Manual, 2nd ed., Dordrecht:Kluwer Academic Publ., 1995, in Sect, Ringbuch Zentrale Signatur: BT1l-PISBN 0-7923-2731-4; Glick B R, Thompson J E, Methods in Plant MolecularBiology and Biotechnology, Boca Raton: CRC Press, 1993, 360 pp., ISBN0-8493-5164-2).

The transformation of Arabidopsis thaliana by means of Agrobacterium wascarried out by the method of Bechthold et al., 1993 (C.R. Acad. Sci.Ser. III Sci. Vie., 316, 1194-1199). For example, oilseed rape can betransformed by cotyledon or hypocotyl transformation (Moloney etal.(1989) Plant Cell Report 8:238-242; De Block et al.(1989) PlantPhysiol 91: 694-701). The use of antibiotics for the selection ofagrobacteria and plants depends on the binary vector used for thetransformation and the agrobacterial strain. The selection of oilseedrape is usually carried out using kanamycin as-selectable plant marker.

Agrobacterium-mediated gene transfer into linseed (Linum usitatissimum)can be carried out for example using a technique described by Mlynarovaet al. (1994) Plant Cell Report 13:282-285. Soya can be transformed forexample using a technique described in EP-A-0 0424 047 (Pioneer Hi-BredInternational) or in EP-A-0 0397 687, U.S. Pat. No. 5,376,543, U.S. Pat.No. 5,169,770 (University of Toledo).

The transformation of plants using particle bombardment, polyethyleneglycol mediated DNA uptake or via the silicon carbonate fiber techniqueis described, for example, by Freeling and Walbot “The Maize Handbook”(1993) ISBN 3-540-97826-7, Springer Verlag New York).

Example 6 Studying the Expression of a Recombinant Gene Product in aTransformed Organism

The activity of a recombinant gene product in the transformed hostorganism was measured at the transcription and/or translation level.

A suitable method for determining the level of transcription of the gene(which indicates the amount of RNA available for translating the geneproduct) is to carry out a Northern blot as described hereinbelow (forreference see Ausubel et al. (1988) Current Protocols in MolecularBiology, Wiley: N.Y., or the above examples section), where a primerwhich is designed such that it binds to the gene of interest is labeledwith a detectable label (usually a radiolabel or chemiluminescent label)so that, when the total RNA of a culture of the organism is extracted,separated on a gel, transferred to a stable matrix and incubated withthis probe, binding and the extent of binding of the probe indicates thepresence and the amount of mRNA for this gene. This informationindicates the degree of transcription of the transformed gene. Cellulartotal RNA can be prepared from cells, tissues or organs using severalmethods, all of which are known in the art, for example the methodBormann, E. R., et al. (1992) Mol. Microbiol. 6:317-326.

Northern Hybridization:

To carry out the RNA hybridization, 20 μg of total RNA or 1 μg ofpoly(A)+RNA were separated by means of gel electrophoresis in 1.25%strength agarose gels using formaldehyde and following the methoddescribed by Amasino (1986, Anal. Biochem. 152, 304), transferred topositively charged nylon membranes (Hybond N+, Amersham, Brunswick) bycapillary force using 10×SSC, immobilized by UV light and prehybridizedfor 3 hours at 68° C. using hybridization buffer (10% dextran sulfatew/v, 1 M NaCl, 1% SDS, 100 mg herring sperm DNA). The DNA probe waslabeled with the Highprime DNA labeling kit (Roche, Mannheim, Germany)during the prehybridization step, using alpha-³²P-dCTP (AmershamPharmacia, Brunswick, Germany). Hybridization was carried out overnightat 68° C. after addition of the labeled DNA probe in the same buffer.The wash steps were carried out twice for 15 minutes using 2×SSC andtwice for 30 minutes using 1×SSC, 1% SDS, at 68° C. The sealed filterswere exposed at −70° C. for a period of 1 to 14 days.

To study the presence or the relative amount of protein translated fromthis mRNA, standard techniques such as a Western blot may be employed(see, for example, Ausubel et al. (1988) Current Protocols in MolecularBiology, Wiley: N.Y.). In this method, the cellular total proteins areextracted, separated by means of gel electrophoresis, transferred to amatrix like nitrocellulose and incubated with a probe such as anantibody which binds specifically to the desired protein. This probe isusually provided with a chemiluminescent or colorimetric label which canbe detected readily. The presence and the amount of the label observedindicates the presence and the amount of the desired mutated proteinwhich is present in the cell.

Example 7 Analysis of the Effect of the Recombinant Proteins on theProduction of the Desired Product

The effect of genetic modification in plants, fungi, algae, ciliates oron the production of a desired compound (such as a fatty acid) can bedetermined by growing the modified microorganisms or the modified plantunder suitable conditions (as described above) and examining the mediumand/or the cellular components for increased production of the desiredproduct (i.e. lipids or a fatty acid). These analytical techniques areknown to the skilled worker and comprise spectroscopy, thin-layerchromatography, various staining methods, enzymatic and microbiologicalmethods, and analytical chromatography such as high-performance liquidchromatography (see, for example, Ullmann, Encyclopedia of IndustrialChemistry, vol. A2, pp. 89-90 and pp. 443-613, VCH: Weinheim (1985);Fallon A et al. (1987) “Applications of HPLC in Biochemistry” in:Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17;Rehm et al. (1993) Biotechnology, vol. 3, chapter III: “Product recoveryand purification”, pp. 469-714, VCH: Weinheim; Belter P A et al. (1988)Bioseparations: downstream processing for Biotechnology, John Wiley andSons; Kennedy J F und Cabral J M S (1992) Recovery processes forbiological Materials, John Wiley and Sons; Shaeiwitz J A and Henry J D(1988) Biochemical Separations, in: Ullmann's Encyclopedia of IndustrialChemistry, vol. B3; chapter 11, p. 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, NoyesPublications).

In addition to the abovementioned methods, plant lipids are extractedfrom plant material as described by Cahoon et al. (1999) Proc. Natl.Acad. Sci. USA 96 (22):12935-12940, and Browse et al. (1986) AnalyticBiochemistry 152:141-145. Qualitative and quantitative lipid or fattyacid analysis is described by Christie, William W., Advances in LipidMethodology, Ayr/Scotland: Oily Press (Oily Press Lipid Library; 2);Christie, William W., Gas Chromatography and Lipids. A PracticalGuide—Ayr, Scotland: Oily Press, 1989, Repr. 1992, IX, 307 pp. (OilyPress Lipid Library; 1); “Progress in Lipid Research, Oxford: PergamonPress, 1 (1952)-16 (1977) under the title: Progress in the Chemistry ofFats and Other Lipids CODEN.

In addition to measuring the end product of the fermentation, it is alsopossible to analyze other components of the metabolic pathways which areused for producing the desired compound, such as intermediates andsecondary products, in order to determine the overall efficacy of theproduction of the compound. The analytical methods encompassmeasurements of the nutrient quantities in the medium (for examplesugars, carbohydrates, nitrogen sources, phosphate and other ions),measurements of the biomass compositions and of the growth, analysis ofthe production of customary metabolites of biosynthetic pathways, andmeasurements of gases produced during fermentation. Standard methods forthese measurements are described in Applied Microbial Physiology; APractical Approach, P. M. Rhodes and P. F. Stanbury, ed., IRL Press, pp.103-129; 131-163 and 165-192 (ISBN: 0199635773) and references citedtherein.

One example is the analysis of fatty acids (abbreviations: FAME, fattyacid methyl esters; GC-MS, gas-liquid chromatography/mass spectrometry;TAG, triacylglycerol; TLC, thin-layer chromatography).

Unambiguous proof for the presence of fatty acid products can beobtained by analyzing recombinant organisms by analytical standardmethods: GC, GC-MS or TLC, as described variously by Christie and thereferences cited therein (1997, in: Advances on Lipid Methodology,fourth edition: Christie, Oily Press, Dundee, 119-169; 1998,Gaschromatographie-Massenspektrometrie-Verfahren[gas-chromatographic/mass-spectrometric methods], Lipide 33:343-353).

The material to be analyzed can be disrupted by sonication, milling inthe glass mill, liquid nitrogen and milling or other applicable methods.After disruption, the material must be centrifuged. The sediment isresuspended in distilled water, heated for 10 minutes at 100° C., cooledon ice and recentrifuged, followed by extraction in 0.5 M sulfuric acidin methanol with 2% dimethoxypropane for 1 hour at 90° C., which giveshydrolyzed oil and lipid compounds, which give transmethylated lipids.These fatty acid methyl esters are extracted in petroleum ether andfinally subjected to GC analysis using a capillary column (Chrompack,WCOT Fused Silica, CP-Wax-52 CB, 25 mm, 0.32 mm) at a temperaturegradient of between 170° C. and 240° C. for 20 minutes and for 5 minutesat 240° C. The identity of the fatty acid methyl esters obtained must bedefined using standards which are available from commercial sources(i.e. Sigma).

The following protocol was used for the quantitative oil analysis of theArabidopsis plants transformed with the Gpd1 gene:

Lipid extraction from the seeds is carried out by the method of Bligh &Dyer (1959) Can J Biochem Physiol 37:911. To this end, 5 mg ofArabidopsis seeds are weighed into 1.2 ml Qiagen microtubes (Qiagen,Hilden) using a Sartorius (Göttingen) microbalance. The seed material ishomogenized with 500 μl chloroform/methanol (2:1; containsmono-C17-glycerol from Sigma as internal standard) in an MM300 Retschmill from Retsch (Haan) and incubated for 20 minutes at RT. The phaseswere separated after addition of 500 μl 50 mM potassium phosphate bufferpH 7.5. 50 μl are removed from the organic phase, diluted with 1500 μlof chloroform, and 5 μl are applied to Chromarods SIII capillaries fromIatroscan (SKS, Bechenheim). After application of the samples, they areseparated in a first step for 15 mins in a thin-layer chamber saturatedwith 6:2:2 chloroform: methanol: toluene. After the time has elapsed,the capillaries are dried for 4 minutes at room temperature and thenplaced for 22 minutes into a thin-layer chamber saturated with 7:3n-hexane:diethyl ether. After a further drying step for 4 minutes atroom temperature, the samples are analyzed in an Iatroscan MK-5 (SKS,Bechenheim) following the method of Fraser & Taggart, 1988 J.Chromatogr. 439:404. The following parameters were set for themeasurements: slice width 50 msec, threshold 20 mV, noise 30, skim ratio0. The data were quantified with reference to the internal standardmono-C17-glycerol (Sigma) and a calibration curve established withtri-C17-glycerol (Sigma), using the program ChromStar (SKS,Beichenheim).

T2 seeds of several independent transgenic lines with the constructspSUN-USP-gpd1 or pGPTV-gpd1 were analyzed to determine the oil contentsquantitatively. Three independent extractions were carried out with theseed pools of each line, and the extracts were measured independently.The three independent measurements were used to calculate the mean andthe standard deviation.

The result of the measurements for the lines with the constructpGPTV-gpd1 showed a significantly higher oil content in several (10)transgenic lines (FIG. 1) compared to the measurements of 10 wild-typeplants. Similar oil contents are measured for the constructpSUN-USP-gpd1 (not shown).

The average oil content of the above lines is 34.86±1.56%, while theaverage of the wild-type plants is 27.75±2.64%. This corresponds to anabsolute increase in the oil content of 7.1% (relative: 25.6%).

To verify the heritability of the gdpl effect (increased oil content),T2 seeds from the lines with increased oil contents and from lines withunchanged oil contents were planted. In each case 6 plants per line wereplanted out and the seeds were analyzed for oil content and enzymeactivity. The oil content was determined by the methodology describedabove. The data obtained are shown in FIG. 2. Col-0 and C24 Arabidopsisecotypes act as controls. C24 is an ecotype which is distinguished by ahigher oil content than Col-0. It was possible to characterize lineswhose oil contents exceeds that of Col-0. The heritability of theincreased oil content as the effect of the expression of the gdp1 geneswas thus demonstrated.

Example 8 Determination of glycerol-3-phosphate dehydrogenase Activity

A further aim was the demonstration of the direct effect of the enzymein the transgenic plants, in addition to the increased oil content. Todetermine the glycerol-3-phosphate dehydrogenase activity, twoArabidopsis seed pods were harvested per plant and extracted by themethod of Geigenberger and Stitt ((1993) Planta 189:329-339). To thisend, the pods were ground in a mortar under liquid nitrogen and taken upin 200 μl 50 mM HEPES pH 7.4 5 mM 20 MgCl₂, 1 mM EDTA, 1 mM EGTA, 5 mMDTT, 0.1% (w/w) of bovine serum albumin, 2 mM benzamidine, 2 mMamino-n-caproic acid, 0.5 mM phenylmethylsulphonyl, 0.1% Triton X-100and 10% (w/w) glycerol and spun down for 5 minutes, and the supernatantwas divided into aliquots. The production of G3P (glycerol-3-phosphate)from the substrates DHAP (dihydroxyacetone phosphate) and NADH wasmeasured to determine the G3PDH activity. To this end, the oxidation ofNADH was monitored at 340 nm.

The reaction mixture for the activity determination contained 50 mMHEPES pH 7.4, 4 mM DHAP, 0.2 mM NADH and 10 μl of the extraction mix infinal volume of 100 μl. After incubation for 30 minutes at roomtemperature, the reaction was stopped by heating (20 min, 95° C.). Inthe control reaction, the reaction was stopped immediately by heating.

Glycerol-3-phosphate “cycling assay”: 10 μl of the reaction mixture wereadded to 45 μl of a solution comprising 200 mM Tricin, MgCl₂ ₅ mM (pH8.5) and heated (20 min, 95° C.) to destroy remaining DHAP. Thesupernatant was transferred into a 96-well microtiter plate, treatedwith 45 μl of a mixture comprising 2 units G3Pox, 130 units catalase,0.4 unit G3PDH and 0.12 μmol NADH. The reaciton was carried out at 30°C. and the resulting absorption monitored at 340 nm in an Anthos htIImicroplate reader. Reaction rates were calculated on the basis of thedecrease in absorption in (mOD*min-1) using the Biolise software (gibonY et al. (2002) Plant J 30(2):221-235).

The enzyme activity in the transgenic lines #11, #21, #41 and #67 issignificantly higher than in control plants (FIG. 3). The plants withincreased oil contents correlate with plants with increased enzymeactivites. It was thus demonstrated that the increased oil content canbe attributed to the increased conversion of DHAP into G3P, theprecursor of oil synthesis.

1. A method of increasing total oil content in a plant organism or atissue, organ, part, cell or propagation material thereof, comprising:expressing a transgenic yeast glycerol-3-phosphate dehydrogenase in saidplant organism or in said tissue, organ, part, cell or propagationmaterial thereof; and selecting the plant organism or the tissue, organ,part, cell or propagation material thereof in which the total oilcontent in said plant organism or in said tissue, organ, part, cell orpropagation material thereof is increased in comparison with acorresponding plant organism or a tissue organ part, cell or propagationmaterial thereof that is not expressing the transgenic yeastglycerol-3-phosphate dehydrogenase.
 2. The method of claim 1, whereinthe glycerol-3-phosphate dehydrogenase is derived from a yeast selectedfrom the genera consisting of Cryptacoccus, Torulopsis, Pityrosporum,Brettanomyces, Candida, Kloeckera, Trigonopsis, Trichosporon,Rhodotorul, Sporobolomyces, Bullera, Saccharomyces, Debaromyces,Lipomyces, Hansenula, Endomycopsis, Pichia and Hanseniaspora.
 3. Themethod of claim 1, wherein the glycerol-3-phosphate dehydrogenase isderived from a yeast selected from the species consisting ofSaccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha,Schizosaccharomyces pombe, Kluyveromyces lactis, Zygosaccharomycesrouxii, Yarrowia lipolitica, Emericella nidulans, Aspergillus nidulans,Debaryomyces hansenii and Torulaspora hansenii.
 4. The method of claim1, wherein the glycerol-3-phosphate dehydrogenase brings about theconversion of dihydroxyacetone phosphate to glycerol-3-phosphate usingNADH as cosubstrate and has a peptide sequence encompassing at least onesequence motif selected from the group of sequence motifs consisting of:i) GSGNWGT(A/T)IAK; (SEQ ID NO: 22) ii) CG(V/A)LSGAN(L/I/V)AXE(V/I)A;(SEQ ID NO: 26) and iii) (L/V)FXRPYFXV. (SEQ ID NO: 27)


5. The method of claim 1, wherein the glycerol-3-phosphate dehydrogenasebrings about the conversion of dihydroxyacetone phosphate toglycerol-3-phosphate using NADH as cosubstrate and has a peptidesequence encompassing at least one sequence motif selected from thegroup of sequence motifs consisting of: iv) GSGNWGTTIAKV(V/I)AEN; (SEQID NO: 29) v) NT(K/R)HQNVKYLP; (SEQ ID NO: 30) vi) D(I/V)LVFN(I/V)PHQFL;(SEQ ID NO: 31) vii) RA(I/V)SCLKGFE; (SEQ ID NO: 32) viii)CGALSGANLA(P/T)EVA; (SEQ ID NO: 33) ix) LFHRPYFHV; (SEQ ID NO: 34) andx) GLGEII(K/R)FG. (SEQ ID NO: 35)


6. The method of claim 4, wherein the glycerol-3-phosphate dehydrogenaseadditionally encompasses at least one sequence motif selected from thegroup of sequence motifs consisting of: (SEQ ID NO: 23) xi) H(E/Q)NVKYL;(SEQ ID NO: 23) xii) (D/N)(I/V)(L/I)V(F/W)(V/N)(L/I/V)PHQF(V/L/ I); (SEQID NO: 25) xiii) (A/G)(I/V)SC(L/I)KG; and (SEQ ID NO: 28) xiv)G(L/M)(L/G)E(M/I)(I/Q)(R/K/N)F(G/S/A).


7. The method of claim 1, wherein the amino acid sequence of the yeastglycerol-3-phosphate dehydrogenase comprises: the sequence of SEQ ID NO:2, 4, 5, 7, 9, 11, 12, 14, 16, 38 or 40; or a functional equivalent ofsaid sequence, which has an amino acid identity of at least 60% to thesequence of SEQ ID NO:
 2. 8. The method of claim 1, wherein the plant isan oil crop.
 9. The method of claim 1, wherein the total oil content inthe seed of a plant is increased.
 10. A transgenic expression cassettecomprising, a nucleic acid sequence encoding a yeastglycerol-3-phosphate dehydrogenase under the control of a functionalpromoter, wherein the promoter is a seed-specific promoter.
 11. Thetransgenic expression cassette of claim 10, wherein the nucleic acidsequence comprises: the sequence of SEQ ID NO: 1, 3, 6, 8, 10, 13, 15,37 or 39; or a sequence derived from the sequence of SEQ ID NO: 1, 3, 6,8, 10, 13, 15, 37 or 39 in accordance with degeneracy of the geneticcode; or a sequence which has at least 60% identity with the sequence ofSEQ ID NO:
 1. 12. A transgenic vector comprising the expression cassetteof claim
 10. 13. A transgenic plant organism, tissue, organ, part, cellor propagation material thereof, comprising a recombinant yeastglycerol-3-phosphate dehydrogenase.
 14. The transgenic plant organism ofclaim 13, which is an oil crop selected from the group of the oil cropsconsisting of Borago officinalis, Brassica campestris, Brassica napus,Brassica rapa, Cannabis sativa, Carthamus tinctorius, Cocos nucifera,Crambe abyssinica, Cuphea species, Elaeis guinensis, Elaeis oleifera,Glycine max, Gossypium hirsutum, Gossypium barbadense, Gossypiumherbaceum, Helianthus annuus, Linum usitatissimum, Oenothera biennis,Olea europaea, Oryza sativa, Ricinus communis, Sesamum indicum, Triticumspecies, Zea mays, walnut and almond.
 15. A method for the production ofoils, fats, free fatty acids or derivatives thereof, comprisingexpressing a transgenic yeast glycerol-3-phosphate dehydrogenase in aplant organism or tissue, organ, part, cell or propagation materialthereof.
 16. The method of claim 1, wherein the amino acid sequence ofthe yeast glycerol-3-phosphate dehydrogenase contains one or moresequences selected from the group consisting of: SEQ ID NOS. 22-35. 17.The method of claim 1, wherein the total oil content in said plantorganism or in said tissue, organ, part, cell or propagation materialthereof, is increased by at least 10%.
 18. The method of claim 1,wherein the total oil content in said plant organism or in said tissue,organ, part, cell or propagation material thereof is increased by atleast 25%.
 19. The transgenic plant organism, tissue, organ, part, cellor propagation material thereof of claim 13, wherein the recombinantyeast glycerol-3-phosphate dehydrogenase is a part of an expressioncassette.
 20. The transgenic plant organism, tissue, organ, part, cellor propagation material thereof of claim 13, wherein the recombinantyeast glycerol-3-phosphate dehydrogenase is a part of a vector.
 21. Thetransgenic expression cassette of claim 11, wherein the nucleic acidsequence contains at least 80% identity with the sequence of SEQ IDNO:
 1. 22. A transgenic expression cassette which contains a seedspecific promoter and a nucleic acid sequence which comprises a nucleicacid sequence that encodes: the sequence of SEQ ID NO; 2, 4, 5, 7, 9,11, 12, 14, 16, 38 or 10; or an amino acid which has at least 60%identity to the sequence of SEQ ID NO:
 2. 23. The transgenic expressioncassette of claim 22, wherein the amino acid sequence has at least 80%identity to the sequence of SEQ ID NO:
 2. 24. A transgenic expressioncassette that contains a seed specific promoter and a nucleic acidsequence which encodes one or more of the amino acid sequences selectedfrom the group consisting of: SEQ ID NOS. 22-35.
 25. The transgenicexpression cassette of claim 24, wherein the one or more amino acidsequences comprises at least three of the amino acid sequences.
 26. Thetransgenic expression cassette of claim 24, wherein the one or moreamino acid sequences comprises at least five of the amino acidsequences.